Essential oil emulsion nostril treatment composition

ABSTRACT

The invention pertains to liposomes containing an oil-in-water emulsion of a selected combination of at least ten different essential plant oils produced with phospholipids found in human membranes and methods for treating respiratory diseases, disorders or conditions by administering the liposomes into the nose and respiratory system.

BACKGROUND OF THE INVENTION Field of the Invention

The invention pertains to the fields of pharmacy and medicinespecifically to a liposome composition containing a mixture of tenessential oils for treating respiratory diseases, disorders andconditions.

Description of Related Art

The administration of drugs into the nose is a widely used method fortreating nasal congestion caused by allergies, colds or infections. Themost common drugs nasally administered for these conditions aresympathomimetics including pseudoephedrine or phenylephrine which act asdecongestants and produce local vasoconstriction. However, due topenetration of these locally applied drugs through the nasal mucosa intothe general circulation, persons taking these drugs often experienceundesirable side-effects including irritation of, or permanent damageto, nasal mucosal epithelium, respiratory disorders, central nervoussystem stimulation, trouble sleeping, heart palpitations, blood pressureelevation, and in some cases, such as in patients taking MAO inhibitors,potentially fatal hypertensive crises. Moreover, extended use of suchdrugs, especially topical decongestants, can lead to rhinitismedicamentosa a condition of drug dependence characterized by reboundnasal congestion, upon withdrawal of the decongestant. As a result,over-the-counter sprays with these types of active ingredients mayactually lengthen the duration of common cold or sinusitis symptoms.Consequently, doctors often recommend a limited course of such drugs,such as use of a drug for no more than seven days, to minimizeside-effects.

Other conventional medications and treatments for nasal congestionprovide only minimal symptomatic relief and some also have undesirableside effects or other limitations. Over-the-counter medicationscontaining antihistamines sometimes cause drowsiness and impaircognitive judgment so that driving an automobile or operating otherpower driven machinery can be dangerous. Inhaled corticosteroid nasalsprays provide some relief for allergic rhinitis but do not reduceinflammation caused by acute viral infections or inflammation associatedwith other forms of non-allergic rhinitis such as atrophic rhinitis,vasomotor rhinitis, or hormonally-induced vasomotor instability.Prolonged use of steroid sprays has also been associated with drying,cracking and bleeding of the nasal membranes.

Passive treatments, such as irrigation with isotonic saline mists, arealso widely marketed for purposes of moisturizing the nose and relievingcold and allergy symptoms. Isotonic saline mists add moisture to thenose but provide little overall relief for congestion. For example, USPatent Publication US 20130156871 A1 discloses an isotonic nasal washhaving a pH near 7.0 for alleviating dryness, rhinitis, and sinusitis.However, these more passive sprays and treatments have proved to be lessthan satisfactory at providing the relief sought by sufferers of coldsor allergies or other nasal problems.

Various herbal products such as water extracts of various plants havebeen proposed for treatment of rhinitis. U.S. Pat. No. 6,004,560 Adiscloses a nasal spray for treating a fever or cold which is producedby boiling Bupleurum scorzonerifolium Willd, Radix lastidis Indigoticaand Folium lastidis in water and then mixing the boiled liquid withVitamin C. WO 2000041709 A1 discloses a nasal spray based on a chamomileextract and xylometazoline which has a vasoconstrictive property. USPatent Publication US 20130156868 A1 describes a menthol-based nasalsolution for providing a soothing effect on nasal mucosa. Barbourdescribes treatment of avians for various microbial diseases with anatural eucalyptus/peppermint essential oil-based preparation; Barbouret al., Int J. Appl. Res. in Veterinary Medicine 2011 Vol. 9 No 0.4 pp.317-323. In addition, emulsions of some types of essential oils havebeen used to treat tooth decay, vaginal infections, and protozoaninfection; see U.S. Pat. Nos. 9,855,209; 10,154,958 B1; 9,610,314B1; andWO/2018/024516A1. However, many herbal preparations are administered asa bolus and lack persistence in the nasal mucous requiring frequentrepeated administration.

In view of the limitations and drawbacks of prior nasal preparations,the inventors sought to develop an emulsified composition comprising aselect mixture of several natural oils that provides a broader spectrumof palliative, regenerative, antimicrobial and antiallergic properties,which can be formulated inside biocompatible liposomes for sustainedrelease, and which avoids the side-effects of drugs such sympathomimeticand antihistamine drugs or those associated with administration of alarge bolus of a single herbal ingredient.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention is directed to an emulsion of at least tenselected essential oils that is encapsulated in a liposome. The tenessential oils from oregano, thyme, eucalyptus, lime, frankincense,peppermint, lemon, ginger, sage, and lavender oils in combinationprovide a composition with broad antimicrobial, immunostimulatory andpalliative properties. Moreover, the encapsulating liposome comprisescompounds identical to, or biocompatible with, those found in humanmembranes and provides an excellent way to modulate the release of theten emulsified oils.

Another aspect of the invention is a method for treating a nasal, sinusor respiratory disease, disorder, or condition by administering liposomenanoparticles containing the emulsion of a mixture of the ten essentialoils. For example, a composition comprising the liposomes encapsulatingthe emulsion of ten essential oils can be delivered to the nostrils,sinuses or respiratory system as droplets or as a spray or wash to treatviral, bacterial or fungal infections, allergy, inflammation, dryness,or nasal or sinus congestion.

Another aspect of the invention is a method for producing the liposomalcomposition containing a water-in-oil emulsion of the ten essential oilsby drying liposomal components on an inside of rotating container andslowing adding a material to be encapsulated in a liposome.

The liposome composition, typically in the form of nanoparticles,encapsulating the ten selected essential oils provides a valuable andsafe addition to the presently marketed nasal products due to its widerange of antimicrobial, anti-inflammatory, pain killing, andanti-allergy activities and with its ability to moisturize, enhance andheal nasal, sinus and respiratory system membranes. This liposomecomposition also provides for sustained release of the essential oilcomponents for an extended period of time and is produced usingingredients identical or biocompatible with nasal, sinus and respiratorysystem membranes. The encapsulation of the essential oil emulsion withinthe lipid bilayer of the liposome elongates the essential oil releasetime which is associated with direct delivery of the active ingredientsinto mucosal cells. This slower, prolonged release is associated withimproved anti-inflammatory, antiviral, antibacterial, and anti-allergiceffects, as well as with a moisturizing effect that maintains theintegrity of the nasal epithelial layer.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings below.

FIG. 1 depicts differences between micelles, liposomes and lipidbilayers.

FIG. 2 depicts an embodiment of the liposome nanoparticles of theinvention which contains a ten essential oil-in-water emulsion 100within a liposomal lipid bilayer 200.

DETAILED DESCRIPTION OF THE INVENTION

The technology disclosed herein provides a safe liposome-encapsulatedoil-in-water emulsion of ten selected essential oils for treating nasaland respiratory disease, disorders and conditions. According to theWorld Health Organization, respiratory diseases are leading causes ofdeath and disability in the world with annual deaths from respiratoryailments of about 650,000 people. The liposome composition disclosedherein, which contains ten select emulsified essential oils, can preventupper respiratory symptoms and the infiltration of microbes from theupper to the lower part of the respiratory system. The prevention ofinfiltration of microbes from the upper to lower respiratory system isimportant for preventing or reducing the severity of diseases such thosecaused by SARS-COV-2, which binds to, and invades cells via, cellularreceptors in the lower respiratory system. Each of the 10 essential oilswas selected based on different mode of action due differences in activeingredients and concentrations of active ingredients.

The oil-in-water emulsion encapsulated within the liposome nanoparticlesdisclosed herein contains components having a documented record ofsafety. It is made from a mixture of ten organic essential oils whichare oregano, thyme, eucalyptus, lime, frankincense, peppermint, lemon,ginger, sage, and lavender oils. Preferably, the essential oils areextracted from plants that are organically grown and harvested withoutpesticides or herbicides. The emulsion is made from a mixture of theseten oils in combination with one or more emulsifiers, preferably food orpharmaceutical grade emulsifiers. The liposomal membranes whichencapsulate the emulsion of ten essential oils and the emulsifier can beselected to be of the same nature as molecules that form the membrane ofhuman eukaryotic cells. For example, for preferred embodiments, thecontent of the engineered liposome nanoparticles includes phosphatidylcholine and cholesterol, ingredients that are naturally present in themembrane of all human eukaryotic cells; see Cooper G M, et al., SinauerAssociates. Cell membranes. 2000 Available from: hypertext transferprotocol secure://www.ncbi.nlm.nih.gov/books/NBK9928/ (incorporated byreference).

Essential oils are highly concentrated oils extracted from aromaticplants such as oregano and lavender. About 700 different types of plantscontain useful essential oils, and there are several methods used toextract them—the most common of which is distillation, for example,water, water and steam or steam distillation. However, other methodsinclude cold pressing, expeller pressing, and solvent extraction, suchas with organic solvents or supercritical CO₂. Additional informationabout essential oils is incorporated by reference to ISO 4720:2009 whichlists botanical names of plants used for the production of essentialoils, together with the common names of the essential oils in Englishand French; and to ISO 4720:2009 which includes an alphabetical index ofcommon names of essential oils in English and French.

High doses of a single oil can cause undesired side effects and arelimited in antimicrobial spectrum and other functional activities. Oneadvantage of including a blend of all ten essential oils is to use eachoil in a lower concentration to avoid mucosal irritation and sneezingwhile benefiting from the cumulative healing power of the componentoils. In addition, the spectrum of active components in each of the tenoils is very different thus their combination will provide a broadspectrum of molecules for prevention and treatment different infectingagents targeting the upper respiratory system and consequently preventthe infiltration of the infection to lower part of the respiratorysystem, mainly to lungs, thus controlling serious pneumonia conditions.Moreover, the ten essential oils disclosed herein exhibit differentimmunological and healing mechanisms on upper respiratory systemailments, including antiviral, antibacterial, anti-inflammatory, andimmunomodulatory properties, stimulatory effects on cell-mediatedimmunity, anti-cough effects, anti-irritation effects, inhibition ofmicrobial colonization, and antioxidant effects. Accordingly, tendifferent essential oils were selected for incorporation into theoil-in-water emulsion disclosed herein to reduce the concentration ofeach oil and avoid side effects of higher dosages of a single type ofoil as well as to provide additive or synergistic antimicrobial andother therapeutic properties.

These ten essential oils are oregano, thyme, eucalyptus, lime,frankincense, peppermint, lemon, ginger, sage, and lavender oils.Typically, equal volumes (10 vol. %) of each oil are used to produce themixture of essential oils used to produce an essential oil-in-wateremulsion. In some embodiments, the amounts of each oil may range between5, 10, 15 or 20 vol. % based on the total content of essential oils. Thecontent of a mixture of the ten essential oils in the oil-in-wateremulsion may be selected so as to provide a stable emulsion. In someembodiments, the mixed oil (all ten oils) ranges from 1 to 10 vol/vol %based on the total volume of the emulsion. Preferably, the mixed oilcontent ranges from about 1 to about 3 vol/vol. %.

The essential oil of oregano (Lamiaceae) was selected based onrecognition of its antiviral and antibacterial properties. This oil maybe obtained as described by, and incorporated by reference to, Bozin, etal., J. Agric. Food Chem. 54(5), 1822-1828, 2006.

The essential oil of thyme (Thymus) was selected for itsanti-inflammatory and immunomodulatory properties. The oil may beobtained as described by, and incorporated by reference to,Fachini-Queiroz, et al., Evidence-Based Complement Alternat Med. 2012,657026, 2012

Eucalyptus (Eucalyptus) essential oil was selected for its stimulatoryeffect on innate cell-mediated immune response, resulting inimmunomodulation and antimicrobial effects. It may be acquired orproduced as described by, and incorporated by reference to, Sarafino, etal., BMC Immunol. 9, 17, 2008.

Lime oil was selected for its wide spectrum-antimicrobial effect. Thisessential oil may be sourced as described by and incorporated byreference to Prabuseenivasan, S., et al., BMC Complement Altern Med. 6,39, 2006.

Essential oil of frankincense (Boswellia). There are five main speciesof Boswellia that produce true frankincense with anti-cough properties.This essential oil may be acquired or produced as described by, andincorporated by reference to, Journal of Chromatography A. 1261, 158-63,2012.

The essential oil of peppermint, was included in the ten essential oilmixture because spray application of peppermint oil was found to resultin significant and immediate improvement in symptoms of upperrespiratory ailments. This essential oil may be acquired or produced asdescribed by, and incorporated by reference to, Ben-Arye, et al.,Evidence Based Complement Alternat Med. 2011, 690346, 2011.

Lemon oil was selected based on its antimicrobial activity againstrespiratory pathogens like Klebsiella pneumonia. This essential oil maybe acquired or produced as described by, and incorporated by referenceto, Mose, et al., Arzneimittel-Forschung/Drug Research. 7, 687-692,1957. Lemon essential oil is typically composed mainly of two majorchemicals namely, (3-Pinene (25.44%), and Limonene (39.74%), and minorcomponents of Linalool (2.16%), α-Terpineol (7.30%), linalyl acetate(3.01%), Acetate geranyl (3.03%), Nerolidol (6.91%), Acetate neryl(1.74%) and Farnesol (4.28%).

Ginger oil was selected on the bases of its anti-oxidative andanti-inflammatory effects. This essential oil may be acquired orproduced as described by, and incorporated by reference to, Mashhadi, etal., Int J Prev Med. 4 (Suppl 1), S36-S42, 2013. One example of gingerrhizomes essential oil comprised sesquiterpenes (66.66%) andmonoterpenes (17.28%), and lower concentrations of aliphatic compounds(13.58%). The predominant sesquiterpene is zingiberene (46.71%) followedby valencene (7.61%), β-funebrene (3.09%) and selina-4(14),7(11)-diene(1.03%). The major monoterpenes are citronellyl n-butyrate (19.34%),3-phellandrene (3.70%), camphene (2.59%) and α-pinene (1.09%).

Essential oil of sage (Salvia officinalis) was selected for itsantioxidant and antimicrobial activities. This essential oil may beacquired or produced as described by, and incorporated by reference to,Alizadeh, et al. Advances in Environmental Biology 1, 221-226, 2012.Salvia officinalis essential oil is composed of two major chemicalsnamely, cis-thujone (18-43 wt. %) and camphor (4.5-24.5 wt. %),associated with the following minor components of cineole (5.5-13 wt.%), humulene (0-12 wt. %), trans-thujone (3-8.5 wt. %), camphene (1.5-7wt. %), pinene (1-6.5 wt. %), limonene (0.5-3 wt. %), bornyl acetate(maximum of 2.5 wt. %) and linalool (maximum of 1 wt. %).

The essential oil extracted from lavender (Lavandula officinalis Chaix.)showed strong antibacterial and antifungal properties. This essentialoil may be acquired or produced as described by, and incorporated byreference to, Lodhia, et al. Indian Journal of Pharmaceutical Sciences,71(2),134-136, 2009. Lavendula officinalis essential oil has 78compounds with two major constituents linalool and linalyl acetate atconcentrations ranging between 10, 15, 20, 25, 30, 35 or 40 wt % andintermediate concentrations of geraniol, β-caryophyllene and lavandulylacetate in a ranges between 3, 4, 5 and 6 wt % In one isolate oflavender oil the concentration of linalool is about 30.6 wt. % and theconcentration of linalyl acetate is about 14.2 wt. % and theconcentrations of geraniol about 5.3 wt. %, β-caryophyllene about 4.7%,and lavandulyl acetate about 4.4%.

These ten essential oils have been used by humans in folklore medicineand in foods for centuries with proven safety on human body, pendingapplication in specific dose concentrations; Tisserand, R and Young, R.Essential Oil Safety. 2 ed. Edinburgh, UK: Churchill Livingstone,Elsevier, 2014 (incorporated by reference).

The essential oils disclosed herein are typically free of non-oilycomponents found in so-called plant extracts which can include proteinsand water-soluble components. A plant extract may be obtained from anyof the plant parts including the leaves, pulp, seeds, stems, fruit andfruit seeds, as well as from the whole plant. The plant extracttypically contains phytochemicals and/or metabolites that include,without limitation, alkaloids, flavonoids, saponins, carbohydrates,polysaccharides, terpenoids (e.g., monoterpenoids and sesquiterpenoids),steroids, sterols, phenols, tannins, anthraquinones, anthocyanins, aminoacids, proteins, and vitamins.

In some separate embodiments, one, two, three, four or more of the tenessential oils may be omitted or one or more additional oils besides theten specific essential oils described above may be incorporated into anoil-in-water emulsion that is encapsulated into liposomes of liposomenanoparticles. These include additional essential oils selected from oneor more of agar oil, ajwain oil, angelica root oil, anise oil,asafoetida, Balsam of Peru, basil oil, bay oil, Bergamot oil, blackpepper, birch oil, camphor, cannabis flower, caraway oil, cardamom seedoil, carrot seed oil, cedarwood oil, chamomile oil, calamus Root,cinnamon oil, cinnamon cassia, cistus species, citron, citronella oil,clary sage, clove leaf oil, coffee, coriander, costmary oil, costusroot, cranberry seed oil, cubeb, cumin oil/black seed oil, cypress,cypriol, curry leaf, davana oil, dill oil, elecampane, fennel seed oil,fenugreek oil, fir, galangal, galbanum, geranium oil, goldenrod,grapefruit oil, henna oil, helichrysum, hickory nut oil, horseradishoil, hyssop, Idaho tansy, jasmine oil, juniper berry oil, Laurusnobilis, ledum, lemongrass, Litsea cubeba oil, linaloe, mandarin,marjoram, melaleuca, Melissa oil, Mentha arvensis oil/mint oil, moringaoil, mountain savory, mugwort oil, mustard oil, myrrh oil, myrtle, neemoil, Neem Tree Oil, neroli, orange oil, orris oil, Palo Santo, parsleyoil, Patchouli oil, Perilla essential oil, peppermint oil, petitgrain,pine oil, Ravensara, Red Cedar, Roman Chamomile, rose oil, rosehip oil,rosewood oil, sandalwood oil, sassafras oil, savory oil, Schisandra oil,spearmint oil, spikenard, spruce, star anise oil, tangerine, tarragonoil, tea tree oil, Tsuga, turmeric, valerian, vetiver oil, Western RedCedar, wintergreen, ylang-ylang, zedoary, or combinations thereof.

The essential oils described herein may be obtained from commercialsources or isolated from the corresponding plant sources which arepreferably grown under organic conditions without pesticides orherbicides. Organic production of the plants from which the essentialoils may be derived may be certified by Accredited Organic Certificatessuch as those granted by the USDA, see hypertext transfer protocolsecure://www.ams.usda.gov/services/organic-certification/faq-becoming-certifying-agent(last accessed Jun. 5, 2020, incorporated by reference). One commercialsource of USDA certified organic essential oils is Revive EssentialOils, 505 Montgomery Street, Suite 1025, San Francisco, Calif. 94111.Herbs may be harvested at a time of day when plant oil content ishighest, preferably between 10 am to 11:00 am when essential oils arehighly expressed in it. Essential oils may be isolated by various knowntechniques with steam distillation being a preferred method.

Emulsifiers which can be used to emulsify essential oils and water intoan oil-in-water emulsion include polysorbates which are a class ofemulsifiers often used in pharmaceutical, food, and cosmetic products.Polysorbates are oily liquids derived from ethoxylated sorbitan (aderivative of sorbitol) esterified with fatty acids. Some representativepolysorbates are Polysorbate 20 (polyoxyethylene (20) sorbitanmonolaurate), Polysorbate 40 (polyoxyethylene (20) sorbitanmonopalmitate), Polysorbate 60 (polyoxyethylene (20) sorbitanmonostearate) and Polysorbate 80 (polyoxyethylene (20) sorbitanmonooleate). One preferred emulsifier for use in producing theoil-in-water emulsions disclosed herein is polyoxyethylene sorbitanmonolaurate. Other food grade emulsifiers include lecithin, sodiumphosphates and mono- and di-glycerides. In preferred embodiments, theemulsifier is an E20 food grade emulsifier (polyoxyethylene [20]sorbitan monolaurate; Tween 20®) which is incorporated into theessential oil-in-water emulsion in amounts of 0.1 to 8.0 vol/vol %, andpreferably between 1.6-2.0 vol/vol %.

A food grade emulsifier, such as polyoxyethylene sorbitan ester likepolyoxyethylene sorbitan monolaurate (Tween 20®), is able to create ahomogeneous dispersion of the components present in the ten essentialoils and help standardize the antimicrobial, anti-inflammatory,anti-allergy, anti-pain effects of the mixed oils in the emulsion whencontacted with different parts of the nasal or respiratory system. Suchan emulsifier is acceptable as a food additive and may be incorporatedinto the liposome compositions disclosed herein in amounts of 0.1, 0.2,0.5, 1, 2, 5 to 8.0 vol./vol %, and preferably between 1.6-1.8-2.0vol./vol %, is safe based on acceptable daily intake of up to 25 mg/dayof such food grade emulsifiers in foods suggested by WHO in 1974.

An emulsion is a fluid system in which liquid droplets are dispersed ina liquid. One component of the liposome composition disclosed herein isan essential oil-in-water emulsion which is encapsulated in liposomes.Oil droplets in the dispersed phase of the essential oil-in-wateremulsion disclosed herein may range from 10, 50 100, 200, 500, <1,000 nm(nanoemulsion) or 1000 nm (1 μm), 2, 5, 10, 20, 50 to 100 μm(macroemulsion). Preferably, the average diameters of oil droplets ofthe essential oil-in-water emulsions are less than 1,000 nm. The dropletsize in the essential oil-in-water emulsion may be increased ordecreased to modify the pharmacokinetic rate of release of essentialoils when administered to a subject. In some embodiments, thenanoemulsion is stable at room temperature for at least 3, 6, 9 or 12months. In another embodiment, the nanoemulsion releases about 20, 30,40, 50, 60 or 70 wt % of the essential oils over a period of 4, 6, 8,10, or 12 hours when applied to the nostrils or to the nasal mucosa.

In a preferred embodiment, the essential oil-in-water emulsion is passedthrough a colloidal mill at fine stator gap to create a nanoemulsionwith droplet diameter of 3.0 millimicron (3.0/1000 of a micron),resulting in stable emulsion encapsulated in liposome micelles for aperiod of one year, and following application into the nostrils, releaseof around 50% of the essential oil over a period of 12 hrs, followingthe application in the nostrils.

Antioxidants and vitamins. In some embodiments, the essentialoil-in-water emulsion may contain additional ingredients such asantioxidants, vitamins, minerals or pH buffers. Antioxidants includeascorbyl palmitate, BHT, ascorbic acid, sodium ascorbate, monosterolcitrate, tocopherols, propyl gallate, tertiary butylhydroquinone (TBHQ),butylated hydroxyanisole (BHA), vitamin E, or any combination thereof.Antioxidants can stabilize oils in an emulsion and prevent theiroxidation.

Fat soluble vitamins including vitamins A, C (as ascorbyl palmitate), Dand K may optionally be incorporated into the oil-in-water emulsiondisclosed herein. Vitamin A plays a major role in keeping yourrespiratory, intestinal and urinary tract surface linings healthy. Thisnutrient assists in keeping your mucous membranes and skin properlynourished to function as a barrier against harmful viruses and bacteria.Vitamin C is necessary for healthy mucosa and helps reduce inflammation.Vitamin C as ascorbyl palmitate is fat soluble and can absorbed into thecell membrane where ascorbic acid cannot reach. Vitamin D supports thenormal function of the immune system, can benefit the nasal mucosa whichprotects from viral respiratory infections and provides protectionagainst allergies, such as mold allergies. Vitamin D deficiency isassociated with chronic rhinosinusitis (CRS) a chronic inflammatorydisease of the sinuses and nasal mucosa sometimes characterized bypolyposis. Vitamin K is a group of structurally similar, fat-solublevitamins found in foods and in dietary supplements. The human bodyrequires vitamin K for complete synthesis of certain proteins that areneeded for blood coagulation or for controlling binding of calcium inbones and other tissues. It has a role in protecting fine bonystructures in the nasal cavity, such as delicate paranasal sinus bones,from damage from sinus infections and other nasal disorders. Fat solublevitamins may be incorporated in the essential-oil-in-water emulsion inan amount from about 0.001 to about 0.05% by weight.

In some embodiments, the aqueous phase of an oil-in-water emulsion or anaqueous phase containing liposomal nanoparticles may contain mineralcomponents such as zinc (e.g. zinc salts like zinc citrate) or copper(e.g. copper salts like copper gluconate). Zinc and copper are traceminerals that have roles in maintaining the immune system and indiseases plays in which oxidant stress is elevated, such as certainnasal and respiratory diseases. These metals may be present in theliposome composition or in the essential-oil-in-water emulsion in anamount from about 0.001 to about 0.01% by weight.

In some embodiments, the aqueous phase of an oil-in-water emulsion or anaqueous phase containing liposomes or liposome nanoparticles willcontain a buffer. One skilled in the pharmaceutical or medical arts mayselect an appropriate buffer. Some representative buffers includedibasic sodium phosphate, monobasic sodium phosphate, citric acid,sodium citrate, acetic acid, sodium acetate, boric acid, sodiumcarbonate, sodium borate, hydrochloric acid and sodium hydroxide. Theoil-in-water emulsion may be formulated at different pHs depending onthe type of disease, disorder or condition being treated, for example,it may have a pH of <5, 5, 5.5, 6, 6.5, 7, 7.5, 8 or >8. In someembodiments, the oil-in-water emulsion may be adjusted to, or bufferedat the nasal mucosal pH which approximately 5.5-6.5. Often in rhinitisnasal pH increases to pH 7.2-8.3 and the pH of the composition oremulsion may be adjusted to fall within this range, for example, duringhealing of nasal irritation or damage, or may be adjusted to bring thenasal pH down to a more normal value. The oil-in-water emulsion or anaqueous phase containing the liposome nanoparticles may contain anaqueous phase that is isotonic (e.g., about 0.9% salt) with the nasalmucosa. However, in some embodiments, the aqueous phase may be hypo- andhypertonic.

A liposome is a spherical vesicle having at least one lipid bilayer, seeFIG. 1. The liposome can be used as a vehicle for administration ofnutrients and pharmaceutical drugs. Liposomes can be prepared bydisrupting biological membranes (such as by sonication). Preferably, theliposomes disclosed herein have an average diameter of less than 1, 2,or 3 μm and are made by a method comprising drying liposomal componentson an inside of rotating container and slowing adding a material to beencapsulated in the liposome, specifically, an oil-in-water emulsion often essential oils as disclosed herein. Liposomes are typically composedof phospholipids, especially phosphatidylcholine, but may also includeother lipids, such as egg phosphatidylethanolamine, so long as they arecompatible with lipid bilayer structure. In some embodiments, thephospholipids components of the liposomes are phosphatidylcholines,phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols,phosphatidylinositols, phosphatidic acids, mixed chain phospholipids,lysophospholipids, hydrogenated phospholipids, partially hydrogenatedphospholipids, and mixtures thereof.

Preferably, the components used to make the liposomes are selected to besubstantially identical to components present in the human body, such ascells forming the epithelial tissue of the respiratory system.

The engineered liposome nanoparticles disclosed herein are preferablysimilar in structure to the bi-lipid layer of phospholipids that formthe membrane of the targeted mucosal eukaryote cells, thus allowing forfusion of the engineered liposome with the epithelial cells in therespiratory system, and by that allowing for direct delivery of theencapsulated essential oils to act on intracellular infecting microbesand other phagocytized organisms, and on extracellular colonies ofmicrobes present on the surface of the epithelial cells.

Preferably, the liposome nanoparticles disclosed herein lack anyinclusion of a chemical structure, such as a peptide, protein,lipoprotein, glycoproteins, polysaccharides, mucoadhesive proteins, orother components that induce injurious immune reactions via activationor recognition by local immune cells present in the mucosal layer of therespiratory system. It can exclude milk, serum, mucosal, pulmonary,epidermal, and other cellular proteins. Thus it will be substantiallyfree of antigens creating injuries to the mucosal layer contacted withthe liposome nanoparticles. This avoids many problems associated withadministration of non-self antigenic proteins or carbohydrates as wellas denatured or altered autologous proteins or carbohydrates, and helpsavoid undesirable immunological or physiological reactions to theseliposome components when they are administered to a subject.

In one embodiment, the liposome nanoparticles further comprisepolyethylene glycol (PEG) to improve the pharmacokinetics by providing aprotective, steric barrier against interactions with plasma proteins andcells. Other emulsion or liposome stabilizers or preservatives may alsobe included. Preferably, the liposomal nanoparticles are stable for aperiod of at least 0.25, 0.5, 0.75, 1, 1.5 or 2 years, showing no morethan 0.1, 0.2, 0.5 or 1 wt. % separation, and more preferably, no splitor separation of its different aqueous and lipid phases.

Liposome nanoparticle composition. Typically at least 90 or 95 wt. % ofthe essential oil emulsion is encapsulated and the small amount ofunencapsulated essential oil emulsion typically does not produceirritation or injury to the nostrils or sinuses. The composition maycontain about 1, 2, 3, 4, 5 to 10 wt % of surplus emulsified essentialoils, which have not been encapsulated in liposomal nanoparticles andthus fill space between and among the liposomal particles.

In another embodiment, the liposomal nanoparticles disclosed hereincontaining the ten emulsified essential oils may be suspended in anaqueous phase which may contain <0.1, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 95 or >95% liposomes based on the volume orweight of the composition.

Preferably, the composition comprising the liposomal nanoparticles asdisclosed herein is in a liquid form rather than in a form of anemulsion, cream, lotion, gel, ointment, emollient, wax, or othersemisolid or semiliquid form. The composition may have a viscosityranging from 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0 or >2.0 mPa·s.

The liposome nanoparticle composition of the invention is locally ortopically applied and is formulated from ten natural essential oils andomits conventional drugs and medicines such as sympathomimetic andantihistamine drugs. It may be inhaled or washed over a nasal membrane.The ten oils as well as the emulsifier and liposomal components havebeen previously safely used in many products. Thus, its production andsale would not be constrained by many regulations or guidelinespertaining to systemically administered drugs.

Method of administering composition comprising the liposomenanoparticles loaded with an essential oil-in-water emulsion of tenessential oils. The composition may be nasally administered to a subjectin an amount sufficient to reduce the sinus or respiratory irritationssuch as severity of sinus headache associated with inflamed or congestedturbinates or other nasal tissues or for symptomatic relief of sinusheadache and nasal congestion. It may also be administered or relievingsymptoms of sinusidal pain associated with coryza, nasal congestion,allergic rhinitis and sinusitis by the direct application to affectednasal mucosa.

The liposome nanoparticle composition may be administered to treat apatient infected by, or at risk of infection by, viruses such asrhinovirus, respiratory syncytial virus, human parainfluenza viruses,adenovirus, human coronaviruses, human metapneumovirus and otherrespiratory viruses.

It may be administered to a patient infected by, or at risk of infectionby, bacteria such as alpha hemolytic streptococci, beta hemolyticstreptococci, Branhamella catarrhalis, Diptheroids, Haemophilisinfluenzae (beta-lactamase positive and negative), Moraxella species,Pseudomonas aeruginosa, Pseudomonas maltophilia, Serratia marcescens,Staphylococcus aureus, Streptococcus pneumonia and other respiratorybacteria.

It may be administered to a patient infected by, or at risk of infectionby, fungi such as Aspergillus, Mucor and Candida albicans, Fusarium,Curvularia, Cryptococcus, Coccidioides, and Histoplasma.

A dosage regimen may be selected or recommended by a medicalprofessional or self-selected. In a preferred embodiment, two drops(about 100 microliters) of a composition comprising the liposomenanoparticles as disclosed herein is administered to each nostril onceor twice a day during the early signs of either dyspnea, sinus pressure,throat irritation and soreness, stuffy nose due to nasal congestion,resulting from infection by microbes, allergy, or apparent inflammation.An amount of the composition may be selected by a medical professionalor patient based on the underlying condition, size, age or other patientfactors. Typically, an amount of liposome composition used perapplication is about 2 drops (100 microliters) of the liposomalnanoparticle composition per nostril.

A total amount of lipids and other liposome components such as detergentsufficient to encapsulate at least 80, 85, 90, 95 or >95% of theessential-oil-in-water emulsion may be used to produce the liposomecomposition disclosed herein. Preferably, the liposomal nanoparticlecomposition is prepared by incorporating 0.11 to 0.13 g of total lipidsinto a total volume of 100 ml while respecting a molar ratio ofphosphatidyl choline to cholesterol of 7:3, and including a sodiumcholate detergent for dissolving the lipids at a molar ratio ofphosphatidyl choline to sodium cholate of 0.52. In other embodiments,the ratio of phosphatidyl choline to cholesterol may range from 5:3,6:3, 7:3, 8:3, to 9:3 and the of ratio phosphatidyl choline to sodiumcholate from 0.42, 0.52 to 0.62.

The mixture of liposome components is then dialyzed through a membraneof 10,000 cut point for a period of 3 hrs against 500 ml ofphosphate-buffered saline of pH 7.0 to remove excess detergent. Thispreparation will be dried on an inner wall of a 50 ml round flaskattached to a rotary vapor apparatus. The 100 ml essential oil emulsionis added slowly to the dried film on the rotating flask to encapsulateit within the liposomal particles. Smaller diameter liposomes of less orequal to 3 micrometer diameter, suitable for delivery to the upper andlower respiratory passages, are obtained by sonication withnon-continuous pulse, a 50% duty cycle, and a 20 Khz ultrasonicvibration. The complete formulation is filled into sterilized bottles,labeled regulatory or proprietary information, and packaged forshipping.

Devices for delivering the liposome nanoparticle composition disclosedherein to the nose or respiratory tract may be those that areconventionally used in the art for such purposes. These includeinhalers, nebulizers, nose drop bottles, neti pot or other lavagedevice, dropper bottles or any other device suitable for delivering theliposome nanoparticle composition to the nose, sinuses, bronchia, orlungs. The concentration of liposomes in a composition foradministration may be selected based on the needs of the patient, thetype of disease, disorder or condition being treated, as well as on themode of delivery. In some embodiments, the dosage of a compositioncomprising the liposome nanoparticles loaded with the emulsifiedessential oils may range from 50, 100, 200, 500, 1,000 or >1,000 μL. Adosage may be given as needed for symptomatic relief or given 1, 2, 3,4, 5, 6 or more times per day for a period of <1, 2, 3, 4, 5, 6 or >6weeks. A given dose may contain <1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20or >20 μL of the emulsion of ten essential oils or 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2 or >2 μL of the essential oils per se perdose.

Additional embodiments of this technology include, but are not limitedto the following.

One embodiment of the invention is a composition comprising, consistingessentially of, or consisting of liposomes containing an oil-in-wateremulsion comprising at least one pharmaceutically acceptable emulsifierand a mixture of essential plant oils, wherein said mixture comprisesoils of oregano, thyme, eucalyptus, lime, frankincense, peppermint,lemon, ginger, sage, and lavender. Preferably. the essential oilsconsist of those of oregano, thyme, eucalyptus, lime, frankincense,peppermint, lemon, ginger, sage, and lavender, wherein said oil-in-wateremulsion is in a form of liposomes.

Typically, the liposome composition, or liposome nanoparticlecomposition, is in liquid form, such as in droplet form or in a form ofa wash though in some embodiments it may be applied in a form of a gelor other viscous preparation or in a desiccated or concentrated form forreconstitution prior to use.

Advantageously, the emulsifier may be selected to be a food gradeemulsifier that is already used in foods and pharmaceuticals and whichhas been demonstrated to be safe, such as polyoxyethylene sorbitanmonolaurate. The content of the emulsifier is selected to provide auniform emulsion of the ten essential oils. In some embodiments, thecontent ranges from 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5, 7, or 8vol/vol % based on the total volume of the essential oils, emulsifierand water.

The liposomes or liposomal membranes which encapsulate the emulsion often essential oils may be produced from components known in the art andpreferably from components natural to or found in the human body, suchas phosphatidyl choline and cholesterol.

Liposomes typically comprise a detergent, such as a sodium cholatedetergent. Excess detergent may be removed, for example, by dialysisagainst saline, PBS or another physiologically acceptable buffer, afterformation or isolation of the liposomes.

In one embodiment, the liposomes comprise phosphatidyl choline,cholesterol, and a sodium cholate detergent; wherein the molar ratio ofphosphatidyl choline to cholesterol ranges from 8:3 to 6:3 and whereinthe molar ratio of phosphatidyl choline to sodium cholate ranges from0.4 to 0.6. The liposomes in some embodiments may further comprise atleast one phosphoglyceride naturally found in human membranes, otherthan phosphatidyl choline, such as phosphatidyl ethanolamine,phosphatidyl inositol, phosphatidyl serine, or diphosphatidyl glycerol.

Typically, the liposome composition comprises liposomal nanoparticles nomore than 3 microns in average diameter, such as 0.5, 1, 1.5, 2, 2.5, 3microns in diameter. In other embodiments, the liposomal particles mayexceed 3 microns in average diameter.

Preferably, the liposome composition has a pH ranging from 5, 5.5, 6,6.5, 7, 7.5 to 8.

Advantageously the liposome composition disclosed herein containing theten emulsified essential oils can be formulated to be free of irritantsand artificial drugs such as sympathomimetic drugs, antihistamine drugs,corticosteroids, and mast cell stabilizing drugs, as well as proteinsand other potentially immunogenic components such as carbohydrates,glycoproteins, and lipoproteins, especially those exogenous to the humanbody. In one embodiment, the liposomes may further comprise polyethyleneglycol or PEG, a stabilizing agent to prevent degradation of theliposomes during storage, or a preservative, such as an antioxidant thatprevents oxidation of the lipid and other components of the liposome.

Another embodiment of the invention is directed to a method for makingthe liposomal composition described herein containing an emulsion of tenessential oils that comprises mixing essential oils of oregano, thyme,eucalyptus, lime, frankincense, peppermint, lemon, ginger, sage, andlavender to form a mixture of essential oils, incorporating anemulsifier and water into the mixture of essential oils to form anessential oil-in-water emulsion, loading the essential oil-in-wateremulsion into liposomes. The content of essential oils, emulsifier, andliposome components may be selected based on the ranges disclosed hereinfor these compositions and ingredients.

Preferably, this method uses polyoxyethylene sorbitan monolaurate as theemulsifier in an amount ranging from 1 to 3 vol. % based on a totalvolume of the oil-in-water emulsion and the liposome components comprisephosphatidyl choline, cholesterol, and a sodium cholate detergent;wherein the molar ratio of phosphatidyl choline to cholesterol rangesfrom 8:3 to 6:3 and wherein the molar ratio of phosphatidyl choline tosodium cholate ranges from 0.4 to 0.6; and wherein said loadingcomprises producing a dry film of the liposome components on an innersurface of a rotating container, slowly adding the essentialoil-in-water emulsion to the rotating container thereby formingliposomes containing the emulsion, and reducing the average diameters ofthe formed liposomes to 3 μm or less.

Another embodiment of the invention is directed to a method for treatinga respiratory disease, disorder or condition comprising administeringthe liposome composition disclosed herein into the nose or respiratorytissues of a subject in need thereof.

The composition is in a dispensable form, typically, in liquid form fordispensing as a droplet or spray. However, in some embodiments in may bein a more viscous, semisolid or semiliquid form such as in the form of agel.

This method may be used to treat a subject, preferably a human subject,but in some cases other mammalian or avian subjects, having or at riskof acquiring a respiratory disease, disorder or condition. In someembodiments of this method the respiratory disease, disorder orcondition is a nasal or respiratory infection with a virus, bacterium,or fungi, in other embodiments it is allergy, dryness, or irritation.

EXAMPLES Example 1A. Production of Liposomes Containing an EssentialOil-In-Water Emulsion (Small Prep)

An emulsion of the ten essential oils, food grade emulsifier and wateris prepared from 0.2 vol. % of each of the ten essential oils of oreganooil, thyme oil, eucalyptus oil, lime oil, frankincense oil, peppermintoil, lemon oil, ginger oil, sage oil and lavender oil, 1.6 v/v % of theTween 20 emulsifier, and 96.4 vol/vol. % water. These amounts are safebased on an acceptable daily intake of up to 25 mg/day as suggested byWHO in 1974. The food grade emulsifier, polyoxyethylene sorbitanmonooleate monolaurate, is able to create a homogeneous dispersion ofall active ingredients present in the essential oil blend, resulting insimilar antimicrobial, anti-inflammatory, anti-allergy, anti-pain effectat each point of the respiratory tissues.

The preparation of the liposomal nanoparticles for a total volume of 100ml incorporates total lipids of 0.11-0.13 g, while respecting a molarratio of phosphatidyl choline to cholesterol of 7:3, and including asodium cholate detergent for dissolving the lipids at a molar ratio ofphosphatidyl choline to sodium cholate of 0.52. Dialysis is performedthrough a membrane of 10,000 cut point for a period of 3 hrs against 500ml of phosphate-buffered saline of pH 7.0 to remove the excessdetergent. This preparation is dried on the inner wall of a 50 ml roundflask attached to a rotary vapor apparatus.

The 100 ml essential oil emulsion is added slowly to the dried film onthe rotating flask to encapsulate it within the liposomal particles.Smaller diameter liposomes of less or equal to 3 micrometer diameter areobtained by sonication with non-continuous pulse, a 50% duty cycle, anda 20 Khz ultrasonic vibration, to ensure the delivery of the nasalpreparation to upper and lower respiratory passages. The liposomenanoparticle composition is filled in sterilized bottles or suitabledispensing devices, labeled, and packaged for shipping.

Example 1B. Production of Liposomes Containing an Essential Oil-In-WaterEmulsion (Large Prep)

Two hundred milliliters of each of oregano oil, thyme oil, eucalyptusoil, lime oil, frankincense oil, peppermint oil, lemon oil, ginger oil,sage oil, and lavender oil were mixed to form 2 liters of blendedessential oils.

Then, 1.6 L of polyoxyethylene sorbitan monolaurate (E20) wasincorporated into the 2 liters of mixed oils form an oil phase. Thepolyoxyethylene sorbitan monolaurate (E20) emulsifier is used forcreating an essential oil blend-in-water emulsion, is a food grade, usedin many foods, with proven safety on human health. Fruijtier-Polloth,Toxicology. 214, 1-38, 2005, incorporated by reference. Water (96.4 L)is slowly added with gentle propeller mixing to the oil phase to form afinal volume of 100 L on an essential oil-in-water emulsion. Thus, thevolume percent composition of the organic essential oil blend-in-wateremulsion is: water/oregano oil/thyme oil/eucalyptus oil/limeoil/frankincense oil/peppermint oil/lemon oil/ginger oil/sageoil/lavender oil/E20 emulsifier corresponding to96.4/0.2/0.2/0.2/0.2/0.2/0.2/0.2/0.2/0.2/0.2/1.6 (V/V).

The water is added slowly to the oil phase, while mixing gently, using apropeller mixer, to form 100 liters an essential oil-in-water emulsion.

Separately from the production of the essential oil-in-water emulsion, aliposome-forming composition is prepared from phosphatidyl choline,cholesterol and sodium cholate and has a total lipid content rangingfrom 110 to 130 g. These ingredients are mixed in the following ratiosto form the composition: (i) a ratio of 7:3 of phosphatidyl choline tocholesterol, (ii) ratio of 0.52 of phosphatidyl choline to sodiumcholate detergent.

This liposome-forming composition was then dissolved in one liter ofsterile phosphate buffered saline (PBS) and was dialyzed against PBSusing a 10,000 cut point membrane.

The dialyzed composition was then dried on a sterile, clean innersurface of a rotating container with capacity of 150 liters to produce adried film of liposome-forming lipids on the inner surface.

The 100 liters of essential oil blend-in-water emulsion was slowly addedover the dried film of liposome-forming lipids, while the sphericalvessel was rotating to form the liposomes that encapsulate the essentialoil-in-water emulsion within its bilayers.

The resulting liposomes in the 100 L preparation are reduced to adiameter of 3 μm or less using a colloidal mill set at a stator gap of 3μm.

Both the rotor of the mixer and stator of the colloidal mill have smoothsurfaces. The rpm of the mixer and colloidal mill may range between300-3000 rpm; preferably the mill is operated at about 1,000 rpm.

Typically the final volume of the liposome composition is approximatelythe same as that of the oil-in-water emulsion used to make it becausethe thin dried layer of liposomal forming materials on the wall of therotating vessel adds a negligible volume. Thus, after milling andencapsulation within the liposome the volume will be about 100 ml.Preferably, it is not necessary to concentrate the liposomal content ofthe prepared composition before use. The composition may be formulatedwith a known content of liposome-forming materials and dried on theinside wall of the rotating flask and a known amount of essentialoil-in-water emulsion is slowly added to the flask to be encapsulatedwithin the liposome vesicles and is suspended in the aqueous environmentforming its bilayer structure. In other embodiments, the concentrationof liposomes produces as disclosed herein may be adjusted either byconcentration of the liposomes or by dilution, for example, by admixturewith a pharmaceutically acceptable carrier or other active ingredientsto dilute the concentration of liposomes to 5, 10, 20, 30, 40, 50, 60,70, 80, 90 or <100% of their original concentration.

Example 2. Evaluation of Antibacterial Properties of LiposomesContaining Essential Oil-In-Water Emulsion

The nasal mucosa of twenty people, 10 men and 10 women 30-35 years old,are sampled using nasal swabs before and after treatment with liposomescontaining the essential oil-in-water emulsion as described inExample 1. Samples are taken 0 hours (control) and at 6 and 12 hoursafter treatment with 2 drops per nostril (0.1 ml/nostril) of theliposomes.

Each sample is suspended in sterile saline solution, serially diluted,plated, and incubated aerobically. The bacterial count on the plates wasrecorded and compared to control values, see Table 1 below.

TABLE 1 Mean bacterial count in nasal swabs collected before treatmentwith nasal preparation and at three times after treatment Mean Bacterialcount/nasal swab Before After Treatment with Nasal Preparation (Hrs)Gender Treatment 1 6 12 Male (3.0 × 10⁴)^(a, 1) (5.0 × 10³)^(a, 2) (2.7× 10²)^(a, 3) (5.3 × 10)^(a, 4) Female (2.2 × 10⁴)^(a, 1) (3.4 ×10³)^(a, 2) (1.8 × 10)^(a, 3 ) (4.2 × 10)^(a, 4) ^(a)Means in a columnfollowed by the same alphabet superscript are insignificantly differentat P > 0.05. ¹⁻⁴Means in a row followed by different Arabic numericalsuperscripts are significantly different (P < 0.05). Means of the countsat different times were compared statistically by one way ANOVA followedby conservative Tukey's Test, including comparison of each of the twogenders at different times.

Example 3. Targeted Reduction of Nasal Congestion at Different Points inTime in Hospitalized Patients Having Severe Respiratory Symptoms afterAdministration of Liposomes Containing Essential Oil-In-Water Emulsion

The degree of nasal congestion was evaluated by a medical professionaland the amount of nasal secretions over a 1 minute period is measured inten patients experiencing rhinitis or nasal congestion. Patients werethen administered 2 drops per nostril (0.1 ml/nostril) of the liposomepreparation as described by Example 2. The degree of nasal congestionand amount of nasal secretions were measured at 1, 6 and 12 hours afteradministration of the liposomes and compared to pre-treatment values(Table 2). Nasal congestion was evaluated by a score of 0 or 3, where 0indicated no improvement, while 3 was the maximum improvement observed.A score 3 was confidently given by when the person is had no moredyspnea (difficulty or labored breathing). Results are shown by Table 2and show progressive improvement compared to pretreatment values over aperiod of 12 hours.

Mean Score of nasal congestion (score of 0 or 3) Before After Treatmentin real time (hrs) treatment 1 hr 6 hrs 12 hrs 0¹ 1.2² 1.8³ 2.4⁴¹⁻⁴Means in a row followed by different Arabic numerical superscriptsare significantly different (P < 0.05). Means of the scores at differenttimes were compared statistically by One Way ANOVA followed byconservative Tukey's Test.

Example 4. Evaluation of Antibacterial and Antiviral Properties ofLiposomes Containing Essential Oil-In-Water Emulsion

Suspensions of viable Streptococcus spp. and Klebsiella pneumoniabacteria and suspensions of rhinovirus and influenza H9B2 subtype areprepared.

Titrated amounts of the liposome preparation described in Example 1 areadded to

In vitro susceptibility testing of the invented preparation against grampositive and gram negative bacteria, and against Rhino and influenzaviruses.

The essential oil blend-in-water emulsion is put in contact fordifferent times (0, 5, 15, 30 minutes) against streptococcus spp. (grampositive), Klebsiella pneumonia (gram negative), rhinovirus, andinfluenza H9N2 subtype. The inactivation of the 4 test organisms aftereach contact time is recorded.

Example 5. Ames Testing Liposomal Preparation

To evaluate the safety of the liposomal preparation described in Example1, the Ames test is performed according to the procedure described by,and incorporated by reference to, Ames et al, Carcinogens are mutagens:their detection and classification. Environ Health Perspect. 1973December; 6:115-8; Mortelmans and Zeiger, The Ames Salmonella/microsomemutagenicity assay. Mutat Res. 2000 Nov. 20; 455(1-2):29-60.

Briefly, the test organism Salmonella enterica subsp. Enterica serovartyphimurium (ATCC® 29629-Strain Designations: TA 1535), an auxotrophicmutant, is provided by ATCC, Manassas, USA. This auxotrophic isolate hasa mutation in its DNA which prevents it from producing histidine andthus requires histidine for its growth.

Potentially mutagenic substances are identified by contacting them withthis auxotrophic mutant and measuring the frequency of mutations thatrestore the ability of this auxotroph to grown a medium not containinghistidine.

The liposomal preparation described by Example 1 is Ames tested byincorporating it at four different concentrations: 1/100×, 1×, 1.5×, and2×, where X is the concentration of the final liposome preparationapplied into a histidine-free medium used for the Ames test. Eachconcentration (X) is added to double strength histidine-free medium in1:1 ratio. Double strength medium was used to avoid its dilution due toaddition of an invented preparation in 1:1 ratio.

A negative control culture that is not exposed to the liposomalpreparation and positive control cultures containing 20 or 200 nmoleconcentrations of a known mutagenic compound, ethidium bromide, are alsomade.

Consequently, the auxotrophic S. typhimurium is cultured in triplicateonto the solid culture media having the four different concentrations ofliposomes and in the negative (no liposomes) and positive control media.The bacterium is spread over these media with small amount of histidinethat helps in initiation of the growth. However, once this amino acid isdepleted only cells mutated to produce histidine by the liposomepreparation described by Example 1 continue their growth.

A degree of mutagenicity of the liposome preparation is determined bycomparison of the number of colonies appearing on the negative andpositive controls and the number of colonies on the solid mediumcontaining different concentrations of the liposome preparationcontaining the essential oil-in-water emulsion of Example 1.

Results indicating nonsignificant differences in counts between negativecontrol and test plates comprising the 1/100×, 1×, 1.5× and 2×concentrations of the liposome preparation indicate that the preparationis non-mutagenic as shown by the Ames test. A positive result with asignificant increase in colony counts on the positive control platesincorporating 20 and 200 nmol ethidium bromide is expected.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all subranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

1. An essential oil emulsion nostril treatment composition, comprising: liposomal particles containing an oil-in-water emulsion said oil-in-water emulsion consisting of phosphatidyl choline, a sodium cholate detergent, polyoxyethylene sorbitan monolaurate in an amount ranging from 1 to 3 vol. % based on a total volume of the liposomal particle essential oil-in-water emulsion and a mixture of essential plant oils in an amount ranging from 1 to 3 vol. % based on a total volume of the liposomal particle essential oil-in-water emulsion and that is free of water soluble components other than the polyoxyethylene sorbitan monolaurate, wherein said liposomal particles have an average diameter of from 0.5 to 3 microns, wherein said mixture of essential plant oils is a mixture of oils of oregano, thyme, eucalyptus, lime, frankincense, peppermint, lemon, ginger, sage, and lavender, wherein the liposomal particle essential oil-in-water emulsion is substantially free of proteins. 2-7. (canceled)
 8. The essential oil emulsion of claim 1 that is free of sympathomimetic drugs, antihistamine drugs, corticosteroids, and mast cell stabilizing drugs.
 9. (canceled)
 10. The essential oil emulsion of claim 1 that is substantially free of carbohydrates, glycoproteins, and lipoproteins.
 11. (canceled)
 12. The essential oil emulsion of claim 1, which is in the form of a droplet.
 13. (canceled)
 14. The essential oil emulsion of claim 1, which has a pH ranging from 5.5 to 7.0. 15-20. (canceled) 